Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Quantitative RT-qPCR primer sequences

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Antibodies

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Western Blot, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Slit and Robo genes are expressed in the Leydig cells of the mouse testis. a RT-qPCR analyses of brain, testicular and purified Leydig cell Slit and Robo mRNA levels (n = 4–6 per group). The relative levels are represented as delta-Ct versus Actb . Data are means ± sem. b SLIT1, -2 and -3 and ROBO1 immunohistochemistry analyses of 8 week-old wild-type mouse testis

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Quantitative RT-PCR, Purification, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 decreases steroidogenesis in Leydig cells in vitro. Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR in MA10 cells treated a for 4 h with vehicle or 1, 5 and 10 µg/ml exogenous SLIT2; b for 2, 4, 8 and 24 h with vehicle or 10 µg/ml SLIT2. c Representative graph of progesterone concentrations (corrected to RNA input) measured in the spent culture media of MA10 cells treated for 8 h with vehicle or 10 µg/ml SLIT2. d Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR and e representative graph of testosterone concentrations (corrected to RNA input) measured in the spent culture media of mouse primary Leydig cell cultures treated for 8 h with vehicle or 10 µg/ml SLIT2. Experiments were performed three times in triplicate. Expression of each transcript was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: In Vitro, Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 decreases CREB phosphorylation. a Quantification of total and phosphorylated CREB, AKT and mTOR protein levels normalized to ACTB in MA10 cells treated for 1 h with vehicle or 10 µg/ml SLIT2. Quantification of total and phosphorylated AKT and CREB protein levels normalized to GAPDH, and expression of Star determined by RT-qPCR, in MA10 cells treated with vehicle or b 100 nM wortmannin, or c 20 µg/ml SC79, for 1 h (protein) or 4 h (mRNA). n = 3–9 samples per group. Expression of Star was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Exogenous SLIT2 alters Leydig cell responsiveness to LH by decreasing Lhcgr expression. a Expression of Lhcgr determined by RT-qPCR in MA10 cells treated for 2, 4, 8 and 24 h with vehicle or 10 µg/ml SLIT2. b Expression of Star , Cyp11a1 , Cyp17a1 and Hsd3b1 determined by RT-qPCR in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 50 ng/ml LH for 4 h. c Quantification of total and phospho-CREB protein levels normalized to GAPDH in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 50 ng/ml LH for 30 min. d Expression of Star and Cyp11a1 determined by RT-qPCR in MA10 cells treated with 10 µg/ml SLIT2 for 24 h, ± 10 µM forskolin for 4 h. n = 3–9 samples per group. Expression of each transcript was normalized to the housekeeping gene Rplp0 . Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.05; ## p < 0.01; ### p < 0.001; NS not significant

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Slit/Robo signaling regulates Leydig cell steroidogenesis

doi: 10.1186/s12964-020-00696-6

Figure Lengend Snippet: Increased testicular steroidogenesis in Robo1- null mice. a Expression of Star , Cyp11a1 and Cyp17a1 determined by RT-qPCR in primary Leydig cells isolated from Robo1 +/+ vs Robo1 Robo1 −/− mice treated for 8 h with vehicle or 10 µg/ml SLIT2. n = 4 samples per group. b Expression of Robo2 determined by RT-qPCR in cultured Leydig cells isolated from Robo1 +/+ and Robo 1 −/− mice. n = 4 samples per group. c Expression of Star , Cyp11a1 , Cyp17a1 , Hsd3b1 and Lhcgr determined by RT-qPCR in testes from four month-old Robo1 +/+ and Robo1 −/− mice. n = 5–7 per group. Expression of each transcript was normalized to the housekeeping gene Rplp0 . d Intra-testicular testosterone concentrations corrected to testis weight measured in two- and four month-old Robo1 +/+ and Robo1 −/− mice, and serum LH levels measured in four month-old mice. n = 5–10 per group. Data are means ± sem; statistical analysis (Student’s T -test): * p < 0.05; ** p < 0.01; *** p < 0.001; # p < 0.05; ## p < 0.01; ### p < 0.001

Article Snippet: The next day, the culture medium was replaced with a serum-free medium, and the cells incubated overnight before treatment with vehicle (PBS) or SLIT recombinant mouse protein (R&D Systems, Minneapolis, MN, USA, SLIT1 #5199-SL, SLIT2 #5444-SL, SLIT3 #9295-SL) at varying concentrations and for varying times.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Cell Culture